Microbacterium Awajiense Sp. Nov., Microbacterium Fluvii Sp. Nov. and Microbacterium Pygmaeum Sp. Nov

Microbacterium awajiense sp. nov., Microbacterium ﬂuvii sp. nov. and Microbacterium pygmaeum sp. nov.

Akiko Kageyama1, Yoshihide Matsuo2, Hiroaki Kasai2, Yoshikazu Shizuri2, Satoshi O¯ mura1;3, and Yoko Takahashi1 1Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan. 2Marine Biotechnology Institute, 3-75-1 Heita, Kamaishi, Iwate 026-0001, Japan. 3The Kitasato Institute, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan. (Received Jan. 15, 2008 / Accepted Feb. 25, 2008 / Published May 16, 2008)

The taxonomic positions of three novel strains isolated from soil, driftwood and sediment samples collected in Japan were investigated based on the results of chemotaxonomic, phenotypic and genotypic characteristics. The strains that we examined were Gram-positive, catalase-positive bacteria with L-ornithine as a diagnostic diamino acid of peptidoglycan. The acyl type of peptidoglycan was N-glycolyl. The major menaquinones were MK-11, -12, -13 and/or -14. Mycolic acids were not detected. The G+C content of the DNA was 68 to 70 mol%. These morphological and chemotaxonomical characters and comparative 16S rDNA analysis of the three isolated strains revealed that they belong to the genus Microbacterium. DNA- DNA relatedness data revealed that the three isolates are three new species of the genus Microbacterium. On the basis of the polyphasic evidence, the isolates should be classiﬁed as novel species of the genus Microbacterium: Microbacterium awajiense sp. nov., Microbacterium ﬂuvii sp. nov. and Microbacterium pygmaeum sp. nov. with the type strains YM13-414T (=MBIC08276T, DSM 18907T), YSL3-15T (=MBIC08277T, DSM 18908T) and KV-490T (=NRRL B-24469T, NBRC 101800T), respectively.

INTRODUCTION EGG medium at 25 C for 30 days. The components of EGG medium are shown in Table 1. The bacterium was The genus Microbacterium was first proposed by then cultured on Marine Broth 2216 (Difco) containing Orla-Jensen (1919) with the type species Microbacterium 1.5% agar after being cultivated for 7–10 days. Strain lacticum, and was emended by Takeuchi & Hatano (1998). YSL3-15T was isolated from driftwood collected in The genus Microbacterium is a member of the family October 2005 at the estuary of Maera River on Iriomote Microbacteriaceae in the order Actinomycetales. In the Island, Japan. The driftwood was crushed in autoclaved present study, strain YM13-414T was isolated from a sedi- artificial seawater. Next, 1/10 diluents of the sample was ment sample, strain YSL3-15T was isolated from driftwood, applied to seawater medium containing 0.1% lignan. and KV-490T was isolated from a soil sample. On the pres- Colonies were isolated after incubation for 1 week at ent phenotypic and chemotaxonomic data strongly indicate 25 C. Strain KV-490T was isolated from a soil sample that these strains belong to the genus Microbacterium. Their collected in the Aoyama Cemetery in Tokyo, Japan. Two phenotypic and phylogenetic characteristics, coupled with grams of soil was suspended in 18 mL of sterile water and data for genomic DNA-DNA relatedness levels, suggest mixed. Soil particles were allowed to sediment, the liquid that these strains should be classified as the novel species phase was diluted to 105 and 100 mL samples were spread Microbacterium awajiense sp. nov., Microbacterium fluvii onto the surface of each plate. GPM agar plates (1.0% sp. nov. and Microbacterium pygmaeum sp. nov. glucose, 0.5% peptone, 0.5% meat extract, 0.3% NaCl and 1.2% agar, pH 7.0) with SOD (300 unit/plate) and catalase MATERIALS AND METHODS (2100 unit/plate) (Takahashi et al., 2003) were used, and these were cultured at 27 C. KV-490T was isolated from Bacterial strains and isolation GPM agar plates with SOD and catalase. Biomass for Strain YM13-414T was isolated from a sediment biochemical and chemotaxonomic characteristics was sample collected from the shore of Yura, Awaji Island, prepared by culturing in TSB broth at 27 C. Japan (depth 20 cm, GPS location: N 3416025.700,E 13457013.800), in September 2004. The samples (0.5– Morphological and biochemical tests 1cm3) were homogenized with a glass rod in 5 mL of Morphological observation under a scanning electron sterile seawater. The homogenate (50 mL) was cultured on microscope (model JSM-5600; JEOL) was performed using

Corresponding author and address: Yoko Takahashi, Kitasato Institute for Life Sciences, Kitasato University, 5-9-1 Shirokane, Minato-ku, Tokyo 108-8642, Japan., phone: +81-3-5791-6133, e-mail: [email protected], fax: +81-3-5791-6133.

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Table 1. Components of EGG medium. EGG medium Metal mix X Metal mix X 250 mL NaCl 500 g ‘‘EGG’’ mix 100 mL MgSO47H2O 180 g Cycloheximide 50 mg CaCl22H2O 2.8 g Gryseoﬂuvin 25 mg KCl 14 g

Nalidixic acid 20 mg Na2HPO412H2O5g Aztreonam 40 mg FeSO47H2O 200 mg Agar 20 g PII metals 600 mL DW 650 mL/pH 7.6 S2 metals 100 mL DW 4300 mL/pH 7.6 EGG mix PII metals

Extract Ehlrich 0.5 g Na2-EDTA 1 g Hurt Infusion Broth 0.2 g H3BO3 1.13 g Gelatin 0.2 g Fe soln. 1 mL of FeCl36H2O (2.42 g/50 mL) Glycerol 0.2 g Mn soln. 1 mL of MnCl24H2O (7.2 g/50 mL) Yeast extract 0.2 g Zn soln. 1 mL of ZnCl2 (0.52 g/50 mL (+HCl)) DW 100 mL/pH 7.6 Co soln. 1 mL of CoCl26H2O (0.2 g/50 mL) DW 996 mL/pH 7.5 S2 metals NaBr 1.28 g

Mo soln 10 mL of Na2MoO42H2O (0.63 g/50 mL) Sr soln. 10 mL of SrCl26H2O (3.04 g/50 mL) Rb soln. 10 mL of RbCl (141.5 mg/50 mL) Li soln. 10 mL of LiCl (0.61 g/50 mL) I soln. 10 mL of KI (6.55 mg/50 mL)

V soln. 10 mL of V2O5 (1.785 mg/50 mL (+NaOH)) DW 940 mL/pH 7.5 cultures grown on GPM agar medium at 27 C for 3 or 6 equipped with a CAPCELL PAK C18 column (Shiseido days. The carbon-assimilation properties of the two strains Co., Ltd.) (Tamaoka et al., 1983). Methyl esters of cellular YM13-414T and KV-490T were determined using Pridham- fatty acids were prepared, and were analyzed by GLC Gottlieb agar medium (Nihon Pharmaceutical Co., Ltd.) (model HP6890; Hewlett-Packard). (Pridham & Gottlieb, 1948), and the carbon-assimilation properties of strain YSL3-15T were determined using ten- G+C content of DNA and DNA-DNA hybridization times diluted Pridham-Gottlieb agar medium supplemented DNA was isolated as described by Saito & Miura with ten-times diluted nutrient agar medium (Difco). NaCl (1963). DNA base composition was determined by HPLC tolerance and the pH and temperature ranges for growth (Tamaoka & Komagata, 1984). Levels of DNA-DNA were determined using 1/5 nutrient agar. The three isolates relatedness were assayed using the method of Ezaki et al. were characterized biochemically using API ZYM (bio- (1989) using photobiotin and a microplates format. Me´rieux) according to the manufacturer’s instructions. 16S rDNA sequencing and phylogenetic analysis Chemotaxonomic tests DNA was prepared by sonication (Yu et al., 2002) or The N-acyl type of muramic acid was determined using using InstaGene matrix (Bio-Rad). 16S rDNA was ampli- the method of Uchida & Aida (1977). Purified cell walls fied by PCR and sequenced with an automatic sequence were obtained using the method of Kawamoto et al. (1981). analyzer (ABI Prism 3130 or 3730; PE Applied Bio- One milligram of purified cell walls was hydrolyzed at systems) using a dye terminator cycle sequencing kit (PE 100 C with 1 mL of 6 N HCl for 16 h. The residue was Applied Biosystems). dissolved in 100 mL of water, and was used for amino acid Species related to the new isolate were identified by analysis by thin layer chromatography (TLC). Mycolic acid performing sequence database searches using the BLAST was assayed using the TLC method of Tomiyasu (1982). program (Altsuchul et al., 1990). Sequence data of related Menaquinones were extracted and purified using the species were retrieved from GenBank. Nucleotide substi- method of Collins et al. (1977), and were then analyzed tution rates (Knuc values) were calculated (Kimura & Ohta, by HPLC (model 802-SC; Jasco) on a chromatograph 1972) and phylogenetic trees were constructed using the

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Table 2. Fatty acid composition (%) of isolated strains. A B C YM13-414T YSL3-15T KV-490T

iso-C15:0 14.04 3.48 1.18 anteiso-C15:0 31.69 48.43 30.45 iso-C16:0 16.65 18.06 7.17 C16:0 1.62 5.91 1.77 iso-C17:0 5.74 1.52 1.82 anteiso-C17:0 28.81 19.58 56.81 C18:0 — 1.48 — Fig. 1. Scanning electron micrographs of cells from 3- or 6-day- old cultures of strains YM13-414T (A), YSL3-15T (B) and KV- 490T (C) grown on GPM agar medium at 27 C. Bar, 2 mm.

Microbacterium fluvii YSL3-15T (AB286028) 41 Microbacterium awajiense YM13-414T (AB286027) Microbacterium deminutum KV-483T (AB234026) 93 Microbacterium pumilum KV-488T (AB234027) Microbacterium flavum YM18-098T (AB286029) Microbacterium lacticum IFO 14135T (AB007415) 45 42 Microbacterium schleiferi DSM20489T (Y17237) Microbacterium koreense JS53-2T (AY962574) Microbacterium pygmaeum KV-490T (AB248875) Microbacterium terregens IFO 12961T (AB004721) T 68 Microbacterium lacus A5E-52 (AB286030) Microbacterium aurum DSM 8600T (Y17229) 64 Microbacterium aoyamense KV-492T (AB234028) Microbacterium terricolae KV-448T (AB234025) M. liquefaciens DSM 20638T (X77444) 0.002 Knuc

Fig. 2. Phylogenetic tree derived from 16S rDNA sequences and created using the neighbor-joining method. Numbers at branching points refer to bootstrap values (1000 resamplings). Only values with >40% are indicated. The tree was unrooted, and Microbacterium liquefaciens was used as an outgroup.

T T neighbor-joining method (Saitou & Nei, 1987). The 414 , anteiso-C15:0 for YSL3-15 and anteiso-C15:0 and T statistical signiﬁcance of the tree topology was evaluated anteiso-C17:0 for KV-490 (Table 2). by bootstrap analysis of sequence data using CLUSTAL We obtained nearly complete 16S rDNA gene sequences W software (Thompson et al., 1994). Sequence similarity of the three isolated strains. Subsequent 16S rDNA-based values were determined by visual comparison and manual phylogenetic analysis demonstrated that the strains be- calculation. longed to the genus Microbacterium. Figure 2 shows the relationship between the three isolated strains and their RESULTS AND DISCUSSION near phylogenetic relatives. YM13-414T and YSL3-15T were related to M. deminutum and M. pumilum, and the The three strains were Gram-positive, aerobic, irregular similarity values of 16S rDNA sequences among these four rods (Fig. 1). The DNA base composition of the three strains ranged from 98.2 to 98.9%. On the other hands, KV- strains was 68 to 70 mol% G+C. The cell wall peptido- 490T was too similar to deduce the interspecies relationship glycans of the three isolates contained L-ornithine as a between type strains of Microbacterium, bootstrap values diagnostic diamino acid. The major menaquinones were in the neighbor-joining tree were not so high. The similarity MK-12, -13 and -14 for YM13-414T, MK-11 and -12 for values of the 16S rDNA sequence between KV-490T and YSL3-15T and MK-11, -12 and -13 for KV-490T. The acyl other Microbacterium species are as follows; M. terregens type of peptidoglycan was N-glycolyl. The major cellular (98.8%), M. lacus (98.8%), M. aurum (98.4%) and fatty acids were anteiso-C15:0 and anteiso-C17:0 for YM13- M. aoyamense (99.1%).

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Table 3. Characteristics of the isolated strains and related Microbacterium species. 12345678 Growth at 37 C+ Growth in NaCl at 5% + + +ND Enzyme assay (APIZYM) Alkaline phosphatase + w w+w w Lipase + + ww+++ Leucine arylamidase + + + + + + + + -Galactosidase +++ -Galactosidase w w ++ + -Glucuronidase ++ -Glucosidase + ++++++ -Glucosidase + + + + + +w -Fucosidase + + G+C content 70 70 68 69 71 69 69 69 Major menaquinones (MK) 12,13,14 11,12 11,12,13 12,13,14 12,13,14 12,13 12,13,14 12,13 1, YM13-414T; 2, YSL3-15T; 3, KV-490T;4,M. deminutum;5,M. pumilum;6,M. lacus;7,M. aoyamense;8,M. terregens. Data are from present study, Kageyama et al. (2006 & 2007). Abbreviations: +, positive; w, weakly positive; , negative; ND, no data.

DNA-DNA hybridization relatedness was determined. lipase (C8), lipase (C14), leucine arylamidase, valine The relatedness value between YM13-414T and YSL3-15T, arylamidase, cystine arylamidase, chymotrypsin, acid and M. deminutum and M. puilum was <23%. The results phosphatase, naphthol-AS-BI-phosphohydrolase, -gluco- showed that these two isolates are independent new species. sidase, -glucosidase, N-acetyl--glucosaminidase and - Species with ornithine in the cell wall and high 16S rDNA fucosidase are detected by the API ZYM enzyme assay; sequence similarity values were used to characterize KV- trypsin, -galactosidase, -glucuronidase and -manno- 490T. Representative values of DNA-DNA relatedness sidase are negative. Weak reaction for -galactosidase is values between KV-490T and M. lacus, M. aoyamense, and detected. The acyl type of the peptidoglycan was N- M. teregens were <21%. These values were well below the glycolyl. The cell wall peptidoglycans contained L-orni- 70% cut-off point for species classification, as recom- thine as a diagnostic diamino acid. The major menaquinone mended by Wayne et al. (1987). These results confirm that is MK-12, -13 and -14. The major cellular fatty acids are the three isolated strains belonged to three independent new anteiso-C17:0 and anteiso-C15:0. The DNA G+C content is species of the genus Microbacterium. 70 mol%. The type strain is YM13-414T (=MBIC08276T, The chemotaxonomic and morphological characteristics DSM 18907T), which was isolated from the sediment sam- of these three isolated strains are consistent with their ple collected from the shore of Yura, Awaji Island, Japan. assignment to the genus Microbacterium (Takeuchi & Hatano, 1998). The phenotypic characters showed that Description of Microbacterium fluvii sp. nov. the isolated strains were distinguished from their nearest Microbacterium fluvii (flu’ vi.i. L. gen. n. fluvii, of a river). phylogenetic neighbors is presented in Table 3. Cells are irregular rods, varying in their cell size from 0.4 Based on the present results, we propose three novel to 0.6 by 0.6 to 1.2 mm. Gram-positive, non-motile, catalase Microbacterium species: Microbacterium awajiense sp. positive, aerobic. Colonies are pale yellow. Growth occurs nov., Microbacterium fluvii sp. nov. and Microbacterium between pH 6 and pH 11, and 16 C and 36 C. In 1/5 pygmaeum sp. nov. Nutrient agar medium, NaCl is tolerated up to 3%. Glucose, galactose, maltose, mannitol, mannose, and rhamnose are Description of Microbacterium awajiense sp. nov. assimilated. Raffinose, sucrose, treharose and xylose are Microbacterium awajiense (awa.ji. en’se. N.L. neut. adj. not assimilated. Esterase (C4), esterase lipase (C8), lipase awajiense, referring to Awaji Island, Hyogo, Japan, where (C14), leucine arylamidase, valine arylamidase, cystine the strain was isolated). arylamidase, trypsin, chymotrypsin, acid phosphatase, Cells are irregular rods, varying in their cell size from 0.5 naphthol-AS-BI-phosphohydrolase, -glucosidase and N- to 0.8 by 0.7 to 1.2 mm. Gram-positive, non-motile, catalase acetyl--glucosaminidase are detected by the API ZYM positive, aerobic. Colonies are light yellow. Growth occurs enzyme assay; -galactosidase, -glucuronidase, -gluco- between pH 6 and pH 11, and 13 C and 38 C. In 1/5 sidase -mannosidase and -fucosidase are negative. Weak Nutrient agar medium, NaCl is tolerated up to 5%. Glucose, reaction for alkaline phosphatase and -galactosidase are galactose, maltose, mannitol, mannose, raffinose, rham- detected. The acyl type of the peptidoglycan was N- nose, trehalose and xylose are assimilated. Sucrose is not glycolyl. The cell wall peptidoglycans contained L-orni- assimilated. Alkaline phosphatase, esterase (C4), esterase thine as a diagnostic diamino acid. The major menaquinone

4 ACTINOMYCETOLOGICA VOL. 22, NO. 1 is MK-11 and -12. The major cellular fatty acid is anteiso- Bacteriol. 39, 224–229. C15:0. The DNA G+C content is 70 mol%. The type strain Kageyama, A., Takahashi, Y., Matsuo, Y., Adachi, K., Kasai, is YSL3-15T (=MBIC08277T, DSM 18908T), which was H., Shizuri, Y. & O¯ mura, S. (2007). Microbacterium flavum isolated from driftwood collected at the estuary of Maera sp. nov. and Microbacterium lacus sp. nov., isolated from River in Iriomote Island, Japan. marine environments. Actinomycetol. 21, 53–58. Kageyama, A., Takahashi, Y. & O¯ mura, S. (2006). Micro- bacterium deminutum sp. nov., Microbacterium pumilum sp. Description of Microbacterium pygmaeum sp. nov. nov. and Microbacterium aoyamense sp. nov. Int. J. Syst. Evol. Microbacterium pygmaeum (pyg.ma’e. um. L. neut. adj. Microbacteriol. 56, 2113–2117. pygmaeum, dwarf). Kawamoto, I., Oka, T. & Nara, T. (1981). Cell wall composition Cells are irregular rods, rods vary in cell size from 0.3 to of Micromonospora olivoasterospora, Micromonospora saga- 0.4 by 0.5 to 0.9 mm. Gram-positive, non-motile, catalase miensis, and related organisms. J. Bacteriol. 146, 527–534. positive, aerobic. Colonies are pale yellow. Growth occurs Kimura, M., & Ohta, T. (1972). On the stochastic model for between pH 5 and pH 9, and 17 C and 31 C. In 1/5 estimation of mutation distance between homologous proteins. Nutrient agar medium, NaCl is tolerated up to 5%. Glucose, J. Mol. Evol. 2, 87–90. galactose, maltose, mannitol, mannose, raffinose, sucrose, Orla-jensen, S. (1919). The Lactic Acid Bacteria. Copenhagen: and trehalose are assimilated, but rhamnose, and xylose are Host & Sons. Pridham, T. G. & Gottlieb, D. (1948). The utilization of carbon not. Esterase (C4), esterase lipase (C8), leucine arylami- compounds by some Actinomycetales as an aid for species dase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, determination. J. Bacteriol. 56, 107–114. -glucosidase, -glucosidase, and N-acetyl--glucosamini- Saito, H. & Miura, K. (1963). Preparation of transforming dase are detected by the API ZYM enzyme assay; deoxyribonucleic acid by phenol treatment, Biophys. Acta 72, phosphatase alkaline, lipase (C4), cystine arylamidase, 619–629. trypsin, chymotrypsin, -galactosidase, -galactosidase, - Saitou, N., & M. Nei. (1987). The neighbor-joining method: a glucuronidase, -mannosidase and -fucosidase are neg- new method for reconstructing phylogenetic trees. Mol. Biol. ative. Weak reaction for valine arylamidase. The acyl Evol. 4, 406–425. type of the peptidoglycan was N-glycolyl. The cell wall Takahashi, Y., Katoh, S., Shikura, N., Tomoda, H. & O¯ mura, S. (2003). peptidoglycans contained L-ornithine as a diagnostic Superoxide dismutase produced by soil bacteria diamino acid. The major menaquinone is MK-11, -12 and increases bacterial colony growth from soil samples. J. Gen. Appl. Microbiol. 49, 263–266. -13. The major cellular fatty acids are anteiso-C and 17:0 Takeuchi, M. & Hatano, K. (1998). Union of the genera anteiso-C15:0. The DNA G+C content is 68 mol%. The type Microbacterium Orla-Jensen and Aureobacterium Collins et al. T T T strain is KV-490 (=NRRL B-24469 , NBRC 101800 ), In a redefined genus Microbacterium. Int. J. Syst. Bacteriol. 48, which was isolated from soil, Aoyamareien, Japan. 739–747. Tamaoka, J., Katayama-Fujimura, Y. & Kuraishi, H. (1983). ACKNOWLEDGEMENTS Analysis of bacterial menaquinone mixtures by high performance liquid chromatography. J. Appl. Bacteriol. 54, 31–36. We are grateful to Dr Jean P. Euze´by (Socie´te´ de Tamaoka, J. & Komagata, K. (1984). Determination of DNA Bacte´riologie Syste´matique et Ve´te´rinaire and Ecole base composition by reversed-phase high-performance liquid Nationale Ve´te´rinaire de Toulouse, France) for his help chromatography, FEMS Microbiol. Lett. 25, 125–128. with the latinization of the species name. This study was Thompson, J. D., D. G. Higgins, & T. J. Gibson. (1994). CLUSTAL W: Improving the sensitivity of progressive multi- supported in part by a Grant of the 21st Century COE ple sequence alignment through sequence weighting, position Program from the Ministry of Education, Culture, Sports, specific gap penalties and weight matrix choice. Nucleic Acids Science and Technology (MEXT), and The JSPS Grant-in- Res. 22, 4673–4680. Aid for Science Research foundation, and New Energy and Tomiyasu, I. (1982). Mycolic acid composition and thermally Industrial Technology Development Organization (NEDO). adaptative changes in Nocardia asteroides. J. Bacteriol. 151, 828–837. REFERENCES Uchida, K. & Aida, K. (1977). Acyl type of bacterial cell wall: its simple identification by a colorimetric method. J. Gen. Appl. Altsuchul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, Microbiol. 23, 249–260. D. J. (1990). Basic local alignment search tool. J. Mol. Biol. Wayne, L. G., Brenner, D. J., Colwell, R. R., Grimont, 215, 403–410. P. A. D., Kandler, O., Krichevsky, M. I., Moore, L. H., Collins, M. D., Pirouz, T., Goodfellow, M. & Minnikin, D. E. Moore, W. E. C., Murray, R. G. E. & other authors (1987). (1977). Distribution of menaquinones in actinomycetes and International Committee on Systematic Bacteriology. Report of corynebacteria. J. Gen. Microbiol. 100, 221–230. the ad hoc committee on reconciliation of approaches to Ezaki, T., Hashimoto, Y. & Yabuuchi, E. (1989). Fluorometric bacterial systematics. Int. J. Syst. Bacteriol. 37, 463–464. deoxyribonucleic acid-deoxyribonucleic acid hybridization in Yu, L., Takahashi, Y., Matsumoto, A., Seino, A., Iwai, Y. & microdilution wells as an alternative to membrane filter O¯ mura, S. (2002). Application of PCR for selection of gram- hybridization in which radioisotopes are used to determine positive bacteria with high DNA G+C content among new genetic relatedness among bacterial strains. Int. J. Syst. isolates. Actinomycetol. 16, 1–5.